Acylated glucagon analogue

ABSTRACT

The invention provides materials and methods for the treatment of obesity and excess weight, diabetes, and other associated metabolic disorders. In particular, the invention provides novel acylated glucagon analog peptides effective in such methods. The peptides may mediate their effect by having increased selectivity for the GLP-1 receptor as compared to human glucagon.

FIELD OF THE INVENTION

The present invention relates to acylated glucagon analogues and theirmedical use, for example in the treatment of obesity and excess weight,diabetes, and other metabolic disorders.

BACKGROUND OF THE INVENTION

Pre-proglucagon is a 158 amino acid precursor polypeptide that isdifferentially processed in the tissues to form a number of structurallyrelated proglucagon-derived peptides, including glucagon (Glu),glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), andoxyntomodulin (OXM). These molecules are involved in a wide variety ofphysiological functions, including glucose homeostasis, insulinsecretion, gastric emptying and intestinal growth, as well as regulationof food intake.

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon. Oxyntomodulin (OXM) is a 37 amino acid peptidewhich includes the complete 29 amino acid sequence of glucagon with anoctapeptide carboxyterminal extension (amino acids 82 to 89 ofpre-proglucagon, and termed “intervening peptide 1” or IP-1. The majorbiologically active fragment of GLP-1 is produced as a 30-amino acid,C-terminally amidated peptide that corresponds to amino acids 98 to 127of pre-proglucagon.

Glucagon helps maintain the level of glucose in the blood by binding toglucagon receptors on hepatocytes, causing the liver to releaseglucose—stored in the form of glycogen—through glycogenolysis. As thesestores become depleted, glucagon stimulates the liver to synthesizeadditional glucose by gluconeogenesis. This glucose is released into thebloodstream, preventing the development of hypoglycemia.

GLP-1 decreases elevated blood glucose levels by improvingglucose-stimulated insulin secretion and promotes weight loss chieflythrough decreasing food intake.

OXM is released into the blood in response to food ingestion and inproportion to meal calorie content. OXM has been shown to suppressappetite and inhibit food intake in humans (Cohen et al, Journal ofEndocrinology and Metabolism, 88, 4696-4701, 2003; WO 2003/022304). Inaddition to those anorectic effects, which are similar to those ofGLP-1, OXM must also affect body weight by another mechanism, since ratstreated with oxyntomodulin show less body weight gain than pair-fed rats(Bloom, Endocrinology 2004, 145, 2687). Treatment of obese rodents withOXM also improves their glucose tolerance (Parlevliet et al, Am JPhysiol Endocrinol Metab, 294, E142-7, 2008) and suppresses body weightgain (WO 2003/022304). OXM activates both the glucagon and the GLP-1receptors with a two-fold higher potency for the glucagon receptor overthe GLP-1 receptor, but is less potent than native glucagon and GLP-1 ontheir respective receptors. Human glucagon is also capable of activatingboth receptors, though with a strong preference for the glucagonreceptor over the GLP-1 receptor. GLP-1 on the other hand is not capableof activating glucagon receptors. The mechanism of action ofoxyntomodulin is not well understood. In particular, it is not knownwhether some of the extrahepatic effects of the hormone are mediatedthrough the GLP-1 and glucagon receptors, or through one or moreunidentified receptors.

Other peptides have been shown to bind and activate both the glucagonand the GLP-1 receptor (Hjort et al, Journal of Biological Chemistry,269, 30121-30124, 1994) and to suppress body weight gain and reduce foodintake (see, for example, WO 2006/134340, WO 2007/100535, WO 2008/10101,WO 2008/152403, WO 2009/155257, WO 2009/155258, WO2010/070252,WO2010/070253, WO2010/070255, WO2010/070251, WO2011/006497,WO2011/160630, WO2011/160633, WO2013/092703, WO2014/041195,PCT/EP2014/072294 and PCT/EP2014/072293.

Obesity is a globally increasing health problem associated with variousdiseases, particularly cardiovascular disease (CVD), type 2 diabetes,obstructive sleep apnea, certain types of cancer, and osteoarthritis. Asa result, obesity has been found to reduce life expectancy. According to2005 projections by the World Health Organization there are 400 millionadults (age >15) classified as obese worldwide. In the US, obesity isnow believed to be the second-leading cause of preventable death aftersmoking.

The rise in obesity drives an increase in diabetes, and approximately90% of people with type 2 diabetes may be classified as obese. There are246 million people worldwide with diabetes, and by 2025 it is estimatedthat 380 million will have diabetes. Many have additional cardiovascularrisk factors, including high/aberrant LDL and triglycerides and low HDL.

SUMMARY OF THE INVENTION

The invention provides a compound having the formulaR¹-H-Aib-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-R²

wherein

R¹ is H (hydrogen), C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; and

R² is OH or NH₂;

or a pharmaceutically acceptable salt or solvate thereof;

The compound may be:H-H-Aib-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH₂

The compounds of the invention may be considered to be glucagonanalogues. References herein to a glucagon analogue peptide should beconstrued as references to a compound of the invention unless thecontext demands otherwise.

Reference to a compound of the invention should be taken to include anypharmaceutically acceptable salt (e.g. an acetate or chloride salt) orsolvate thereof, unless otherwise stated or excluded by context.

The invention provides a composition comprising a compound of theinvention as defined herein (including pharmaceutically acceptable saltsor solvates thereof, as already described) in admixture with a carrier.In preferred embodiments, the composition is a pharmaceuticalcomposition and the carrier is a pharmaceutically acceptable carrier.The compound may be in the form of a pharmaceutically acceptable salt.

The compounds described herein find use, inter alia, in preventingweight gain or promoting weight loss. By “preventing” is meantinhibiting or reducing when compared to the absence of treatment, and isnot necessarily meant to imply complete cessation of weight gain. Thepeptides may cause a decrease in food intake and/or increased energyexpenditure, resulting in the observed effect on body weight.Independently of their effect on body weight, the compounds of theinvention may have a beneficial effect on glucose control and/or oncirculating cholesterol levels, being capable of lowering circulatingLDL levels and increasing HDL/LDL ratio. Thus the compounds of theinvention can be used for direct or indirect therapy of any conditioncaused or characterised by excess body weight, such as the treatmentand/or prevention of obesity, morbid obesity, obesity linkedinflammation, obesity linked gallbladder disease, obesity induced sleepapnea. They may also be used for the improvement of glycaemic control,or for the prevention or treatment of conditions caused or characterisedby inadequate glucose control or dyslipidaemia (e.g. elevated LDL levelsor reduced HDL/LDL ratio), diabetes (especially Type 2 diabetes),metabolic syndrome, hypertension, atherogenic dyslipidemia,atherosclerosis, arteriosclerosis, coronary heart disease, peripheralartery disease, stroke or microvascular disease. Their effects in theseconditions may be as a result of or associated with their effect on bodyweight, or may be independent thereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

The invention also provides the use of a compound of the invention inthe preparation of a medicament for the treatment of a condition asdescribed above.

The compound of the invention may be administered as part of acombination therapy with an agent for treatment of diabetes, obesity,dyslipidaemia or hypertension.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus the compound of the invention can be used in combination with ananti-diabetic agent including but not limited to a biguanide (e.g.metformin), a sulfonylurea, a meglitinide or glinide (e.g. nateglinide),a DPP-IV inhibitor, an SGLT2 inhibitor, a glitazone, a GLP-1 receptoragonist, an SGLT2 inhibitor (i.e. an inhibitor of sodium-glucosetransport, e.g. a gliflozin such as empagliflozin, canagliflozin,dapagliflozin or ipragliflozin), a GPR40 agonist (FFAR1/FFA1 agonist,e.g. fasiglifam), or an insulin or an insulin analogue.

The compound can further be used in combination with an anti-obesityagent including but not limited to a GLP-1 receptor 1 agonist, peptideYY or an analogue thereof, neuropeptide Y (NPY) or an analogue thereof,cannabinoid receptor 1 antagonist, lipase inhibitor, Human prolsletPeptide (HIP), melanocortin receptor 4 agonist, melanin concentratinghormone receptor 1 antagonist, phentermine (alone or in combination withtopiramate), a combination of norepinephrine/dopamine reuptake inhibitorand opioid receptor antagonist (e.g. a combination of bupropion andnaltrexone), Orlistat™, Sibutramine™, CCK, amylin, pramlintide andleptin and analogues thereof, or a serotonergic agent (e.g. lorcaserin).

The compound can further be used in combination with ananti-hypertension agent including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretic, beta-blocker, or calcium channel blocker.

The compound can be used in combination with an anti-dyslipidaemia agentincluding but not limited to a statin, a fibrate, a niacin, a PSCK9(Proprotein convertase subtilisin/kexin type 9) inhibitor and/or acholesterol absorption inhibitor.

Thus the invention further provides a composition or therapeutic kitcomprising a compound of the invention and for example an anti-diabeticagent, anti-obesity agent, anti-hypertension agent or anti-dyslipidaemiaagent as described above. Also provided is such a composition ortherapeutic kit for use in a method of medical treatment, especially fortreatment of a condition as described above.

The compound of the invention may be made by synthetic chemistry.Accordingly the invention provides a method of synthesis of a compoundof the invention.

The invention may also be made by a combination of recombinant andsynthetic methods. The method may comprise expressing a precursorpeptide sequence, optionally purifying the compound thus produced, andadding or modifying one or more amino acids to produce a compound of theinvention. The step of modification may comprise introduction of an Aibresidue (e.g. by modification of a precursor residue), introduction ofthe substituent [17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG at the sidechain of residue 28, and/or modification of one or both terminal groupsR¹ and R², e.g. by introduction of an amide group R² at the freeC-terminus.

The precursor peptide may be expressed from a nucleic acid encoding theprecursor peptide in a cell or a cell-free expression system comprisingsuch a nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, the conventional one letter and threeletter codes for naturally occurring amino acids are used, as well asgenerally accepted abbreviations for other amino acids, including Aib(α-aminoisobutyric acid).

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon and has the sequenceHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr.Oxyntomodulin (OXM) is a 37 amino acid peptide which includes thecomplete 29 amino acid sequence of glucagon with an octapeptidecarboxyterminal extension (amino acids 82 to 89 of pre-proglucagon,having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala and termed“intervening peptide 1” or IP-1; the full sequence of humanoxyntomodulin is thusHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala).The major biologically active fragment of GLP-1 is produced as a30-amino acid, C-terminally amidated peptide that corresponds to aminoacids 98 to 127 of pre-proglucagon.

The term “native glucagon” thus refers to native human glucagon havingthe sequenceH-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH.

Amino acids within the linear sequence of the compounds of the inventioncan be considered to be numbered consecutively from 1 to 29 in theconventional N-terminal to C-terminal direction. Reference to a“position” should be construed accordingly, as should reference topositions within native human glucagon and other molecules.

The residue at position 28 is a lysine residue (Lys) which is conjugatedto a lipophilic substituent by an amide linkage, via its epsilon aminogroup. Without wishing to be bound by any particular theory, it isthought that the substituent binds plasma proteins (e.g. albumin) in theblood stream, thus shielding the compounds of the invention fromenzymatic degradation and thereby enhancing the half-life of thecompounds. It may also modulate the potency of the compound, e.g. withrespect to the glucagon receptor and/or the GLP-1 receptor.

The substituent has the structure:

The substituent is referred to herein by the shorthand notation[17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG. This indicates that it cannominally be considered to be composed of a 17-carboxy-heptadecanoylunit and a hexapeptide spacer having the sequenceglycine-serine-glycine-serine-glycine-glycine, linked by a glutamic acidresidue in “iso” configuration (i.e. linked via its alpha amino group tothe 17-carboxy-heptadecanoyl unit and via its gamma carboxyl group tothe N-terminal end of the hexapeptide spacer).

The presence of the acid group at the end of the fatty chain is believedto enhance the pharmacokinetic properties of the compound, for example,by increasing half life and/or mean residence time, and reducingclearance. The linker may also contribute to these pharmacokineticproperties. Linkers comprising more than one amino acid unit (ormoieties of similar size) may improve pharmacokinetic propertiescompared to those consisting of just one amino acid unit or the like.These properties may enable the compound to be administered lessfrequently than an equivalent compound with the same peptide backbonebut no modification or a different modification (e.g. a substituent withan aliphatic fatty chain lacking a polar group and/or having a shorterlinker moiety).

The term “conjugated” is used here to describe the physical attachmentof one identifiable chemical moiety to another, and the structuralrelationship between such moieties. It should not be taken to imply anyparticular method of synthesis.

The skilled reader will be well aware of suitable techniques that can beused to perform the coupling reactions using general syntheticmethodologies listed e.g. in “Comprehensive Organic Transformations, AGuide to Functional Group Preparations”, 2nd edition, Larock, R. C.;Wiley-VCH: New York, 1999. Such transformations may take place at anysuitable stage during the synthesis process.

Peptide Synthesis

The compounds of the present invention may be manufactured either bystandard synthetic methods, recombinant expression systems, or any otherstate of the art method. Thus the glucagon analogues may be synthesizedin a number of ways, including, for example, a method which comprises:

(a) synthesizing the peptide by means of solid-phase or liquid-phasemethodology, either stepwise or by fragment assembly, and isolation andpurifying of the final peptide product; or

(b) expressing a precursor peptide sequence from a nucleic acidconstruct that encodes the precursor peptide, recovering the expressionproduct, and modifying the precursor peptide to yield a compound of theinvention.

Expression is typically performed from a nucleic acid encoding theprecursor peptide, which may be performed in a cell or a cell-freeexpression system comprising such a nucleic acid.

It is preferred to synthesize the analogues of the invention by means ofsolid-phase or liquid-phase peptide synthesis. In this context,reference is made to WO 98/11125 and, among many others, Fields, G B etal., 2002, “Principles and practice of solid-phase peptide synthesis”.In: Synthetic Peptides (2nd Edition), and the Examples herein.

For recombinant expression, the nucleic acid fragments encoding theprecursor peptide will normally be inserted in suitable vectors to formcloning or expression vectors. The vectors can, depending on purpose andtype of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector. Preferred cloningand expression vectors (plasmid vectors) are capable of autonomousreplication, thereby enabling high copy-numbers for the purposes ofhigh-level expression or high-level replication for subsequent cloning.

In general outline, an expression vector comprises the followingfeatures in the 5′→3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment, optionally a nucleicacid sequence encoding a leader peptide enabling secretion (to theextracellular phase or, where applicable, into the periplasma), thenucleic acid fragment encoding the precursor peptide, and optionally anucleic acid sequence encoding a terminator. They may compriseadditional features such as selectable markers and origins ofreplication. When operating with expression vectors in producer strainsor cell lines it may be preferred that the vector is capable ofintegrating into the host cell genome. The skilled person is veryfamiliar with suitable vectors and is able to design one according totheir specific requirements.

The vectors of the invention are used to transform host cells to producethe precursor peptide. Such transformed cells can be cultured cells orcell lines used for propagation of the nucleic acid fragments andvectors, and/or used for recombinant production of the precursorpeptides.

Preferred transformed cells are micro-organisms such as bacteria [suchas the species Escherichia (e.g. E. coli), Bacillus (e.g. Bacillussubtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g.M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichiapastoris), and protozoans. Alternatively, the transformed cells may bederived from a multicellular organism, i.e. it may be fungal cell, aninsect cell, an algal cell, a plant cell, or an animal cell such as amammalian cell. For the purposes of cloning and/or optimised expressionit is preferred that the transformed cell is capable of replicating thenucleic acid fragment of the invention. Cells expressing the nucleicfragment can be used for small-scale or large-scale preparation of thepeptides of the invention.

When producing the precursor peptide by means of transformed cells, itis convenient, although far from essential, that the expression productis secreted into the culture medium.

Efficacy

Binding of the relevant compounds to GLP-1 or glucagon (Glu) receptorsmay be used as an indication of agonist activity, but in general it ispreferred to use a biological assay which measures intracellularsignalling caused by binding of the compound to the relevant receptor.For example, activation of the glucagon receptor by a glucagon agonistwill stimulate cellular cyclic AMP (cAMP) formation. Similarly,activation of the GLP-1 receptor by a GLP-1 agonist will stimulatecellular cAMP formation. Thus, production of cAMP in suitable cellsexpressing one of these two receptors can be used to monitor therelevant receptor activity. Use of a suitable pair of cell types, eachexpressing one receptor but not the other, can hence be used todetermine agonist activity towards both types of receptor.

The skilled person will be aware of suitable assay formats, and examplesare provided below. The GLP-1 receptor and/or the glucagon receptor mayhave the sequence of the receptors as described in the examples. Forexample, the assays may employ the human glucagon receptor (Glucagon-R)having primary accession number GI:4503947 and/or the humanglucagon-like peptide 1 receptor (GLP-1R) having primary accessionnumber GI:166795283. (in that where sequences of precursor proteins arereferred to, it should of course be understood that assays may make useof the mature protein, lacking the signal sequence).

EC₅₀ values may be used as a numerical measure of agonist potency at agiven receptor. An EC₅₀ value is a measure of the concentration of acompound required to achieve half of that compound's maximal activity ina particular assay. Thus, for example, a compound having EC₅₀[GLP-1]lower than the EC₅₀[GLP-1] of glucagon in a particular assay may beconsidered to have higher GLP-1 receptor agonist potency than glucagon.

The compounds described in this specification are typically GluGLP-1dual agonists, as determined by the observation that they are capable ofstimulating cAMP formation at both the glucagon receptor and the GLP-1receptor. The stimulation of each receptor can be measured inindependent assays and afterwards compared to each other.

By comparing the EC₅₀ value for the GLP-1 receptor (EC₅₀ [GLP-1-R]) withthe EC₅₀ value for the Glucagon receptor, (EC₅₀ [GlucagonR]) for a givencompound, the relative GLP-1R selectivity can be calculated as follows:Relative GLP-1R selectivity [compound]=(EC₅₀ [GLP-1R])/(EC₅₀[Glucagon-R])

The term “EC₅₀” stands for the half maximal Effective Concentration,typically at a particular receptor, or on the level of a particularmarker for receptor function, and can refer to an inhibitory or anantagonistic activity, depending on the specific biochemical context.

Without wishing to be bound by any particular theory, a compound'srelative selectivity may allow its effect on the GLP-1 or glucagonreceptor to be compared directly to its effect on the other receptor.For example, the higher a compound's relative GLP-1 selectivity is, themore effective that compound may be on the GLP-1 receptor as compared tothe glucagon receptor. Typically the results are compared for glucagonand GLP-1 receptors from the same species, e.g. human glucagon and GLP-1receptors, or murine glucagon and GLP-1 receptors.

The compounds of the invention may have a higher relative GLP-1Rselectivity than human glucagon in that for a particular level ofglucagon-R agonist activity, the compound may display a higher level ofGLP-1R agonist activity (i.e. greater potency at the GLP-1 receptor)than glucagon. It will be understood that the absolute potency of aparticular compound at the glucagon and GLP-1 receptors may be higher,lower or approximately equal to that of native human glucagon, as longas the appropriate relative GLP-1R selectivity is achieved.

Nevertheless, the compounds of this invention may have a lower EC₅₀[GLP-1R] than human glucagon. The compounds may have a lowerEC₅₀[GLP-1-R] than glucagon while maintaining an EC₅₀ [Glucagon-R] thatis less than 100-fold higher than that of human glucagon, less than50-fold higher than that of human glucagon, or less than 10-fold higherthan that of human glucagon.

The compounds of the invention may have an EC₅₀ [Glucagon-R] that isless than one hundred-fold that of human glucagon. The compounds mayhave an EC₅₀ [Glucagon-R] that is less than one hundred-fold (e.g. lessthan 50 fold) that of human glucagon and have an EC₅₀ [GLP-1R] that isless than half that of human glucagon, less than a fifth of that ofhuman glucagon, less than a tenth of that of human glucagon, or lessthat a hundredth of human glucagon, e.g. between 0.01 and 0.1 times thatof glucagon, e.g. between 0.01 and 0.05 times that of human glucagon.

The relative GLP-1R selectivity of the compounds may be between 0.05 and20. For example, the compounds may have a relative selectivity of0.05-0.20, 0.1-0.30, 0.2-0.5, 0.3-0.7, or 0.5-1.0; 1.0-2.0, 1.5-3.0,2.0-4.0 or 2.5-5.0; or 0.05-20, 0.075-15, 0.1-10, 0.15-5, 0.75-2.5 or0.9-1.1.

In certain embodiments, it may be desirable that EC₅₀ of any givencompound for the Glucagon-R or GLP-1R (e.g. for the human glucagon orGLP-1 receptor) should be less than 1 nM. In particular, it may bedesirable that EC₅₀ for the GLP-1R (e.g. for the human GLP-1 receptor)should be less than 1 nM.

Therapeutic Uses

The compounds of the invention may provide attractive treatment and/orprevention options for, inter alia, obesity and metabolic diseasesincluding diabetes, as discussed below.

Diabetes comprises a group of metabolic diseases characterized byhyperglycemia resulting from defects in insulin secretion, insulinaction, or both. Acute signs of diabetes include excessive urineproduction, resulting compensatory thirst and increased fluid intake,blurred vision, unexplained weight loss, lethargy, and changes in energymetabolism. The chronic hyperglycemia of diabetes is associated withlong-term damage, dysfunction, and failure of various organs, notablythe eyes, kidneys, nerves, heart and blood vessels. Diabetes isclassified into type 1 diabetes, type 2 diabetes and gestationaldiabetes on the basis on pathogenetic characteristics.

Type 1 diabetes accounts for 5-10% of all diabetes cases and is causedby auto-immune destruction of insulin-secreting pancreatic β-cells.

Type 2 diabetes accounts for 90-95% of diabetes cases and is a result ofa complex set of metabolic disorders. Type 2 diabetes is the consequenceof endogenous insulin production becoming insufficient to maintainplasma glucose levels below the diagnostic thresholds.

Gestational diabetes refers to any degree of glucose intoleranceidentified during pregnancy.

Pre-diabetes includes impaired fasting glucose and impaired glucosetolerance and refers to those states that occur when blood glucoselevels are elevated but below the levels that are established for theclinical diagnosis for diabetes.

A large proportion of people with type 2 diabetes and pre-diabetes areat increased risk of morbidity and mortality due to the high prevalenceof additional metabolic risk factors including abdominal obesity(excessive fat tissue around the abdominal internal organs), atherogenicdyslipidemia (blood fat disorders including high triglycerides, low HDLcholesterol and/or high LDL cholesterol, which foster plaque buildup inartery walls), elevated blood pressure (hypertension) a prothromboticstate (e.g. high fibrinogen or plasminogen activator inhibitor-1 in theblood), and proinflammatory state (e.g., elevated C-reactive protein inthe blood).

Conversely, obesity confers an increased risk of developingpre-diabetes, type 2 diabetes as well as e.g. certain types of cancer,obstructive sleep apnea and gall-blader disease.

Dyslipidaemia is associated with increased risk of cardiovasculardisease. High Density Lipoprotein (HDL) is of clinical importance sincean inverse correlation exists between plasma HDL concentrations and riskof atherosclerotic disease. The majority of cholesterol stored inatherosclerotic plaques originates from LDL and hence elevatedconcentrations Low Density Lipoproteins (LDL) is closely associated withatherosclerosis. The HDL/LDL ratio is a clinical risk indictor foratherosclerosis and coronary atherosclerosis in particular.

Metabolic syndrome is characterized by a group of metabolic risk factorsin one person. They include abdominal obesity (excessive fat tissuearound the abdominal internal organs), atherogenic dyslipidemia (bloodfat disorders including high triglycerides, low HDL cholesterol and/orhigh LDL cholesterol, which foster plaque buildup in artery walls),elevated blood pressure (hypertension), insulin resistance and glucoseintolerance, prothrombotic state (e.g. high fibrinogen or plasminogenactivator inhibitor-1 in the blood), and proinflammatory state (e.g.,elevated C-reactive protein in the blood).

Individuals with the metabolic syndrome are at increased risk ofcoronary heart disease and other diseases related to othermanifestations of arteriosclerosis (e.g., stroke and peripheral vasculardisease). The dominant underlying risk factors for this syndrome appearto be abdominal obesity.

Without wishing to be bound by any particular theory, it is believedthat the compounds of the invention act as dual agonists both on thehuman glucagon-receptor and the human GLP1-receptor, abbreviated here asdual GluGLP-1 agonists. The dual agonist may combine the effect ofglucagon, e.g. on fat metabolism, with the effect of GLP-1, e.g. onblood glucose levels and food intake. They may therefore act toaccelerate elimination of excessive adipose tissue, induce sustainableweight loss, and improve glycaemic control. Dual GluGLP-1 agonists mayalso act to reduce cardiovascular risk factors such as high cholesterol,high LDL-cholesterol or low HDL/LDL cholesterol ratios.

The compounds of the present invention can therefore be used in asubject in need thereof as pharmaceutical agents for preventing weightgain, promoting weight loss, reducing excess body weight or treatingobesity (e.g. by control of appetite, feeding, food intake, calorieintake, and/or energy expenditure), including morbid obesity, as well asassociated diseases and health conditions including but not limited toobesity linked inflammation, obesity linked gallbladder disease andobesity induced sleep apnea.

The compounds may be useful for promoting weight loss or preventingweight gain in subjects affected by conditions characterised byinadequate control of appetite or otherwise over-feeding, such asbinge-eating disorder and Prader-Willi syndrome.

The compounds may be useful for promoting weight loss or preventingweight gain in subjects affected by associated conditions or riskfactors, such as diabetes, dyslipidaemia, hypertension and sleep apnea.

The compounds of the invention may also be used for the improvement ofglycaemic control and for the prevention or treatment of conditionscaused by or associated with impaired glucose control, includingmetabolic syndrome, insulin resistance, glucose intolerance,pre-diabetes, increased fasting glucose, type 2 diabetes, hypertension,atherosclerois, arteriosclerosis, coronary heart disease, peripheralartery disease and stroke, in a subject in need thereof. Some of theseconditions can be associated with obesity. However, the effects of thecompounds of the invention on these conditions may be mediated in wholeor in part via an effect on body weight, or may be independent thereof.

The synergistic effect of dual GluGLP-1 agonists may also result inreduction of cardiovascular risk factors such as high cholesterol andLDL, which may be entirely independent of their effect on body weight.

Thus the invention provides the use of a compound of the invention inthe treatment of a condition as described above, in an individual inneed thereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

The invention also provides the use of a compound of the invention inthe preparation of a medicament for use in a method of treatment of acondition as described above.

In a preferred aspect, the compounds described may be used in treatingdiabetes, esp. type 2 diabetes.

In a specific embodiment, the present invention comprises use of acompound for treating diabetes, esp. type 2 diabetes in an individual inneed thereof.

In a not less preferred aspect, the compounds described may be used inpreventing weight gain or promoting weight loss.

In a specific embodiment, the present invention comprises use of acompound for preventing weight gain or promoting weight loss in anindividual in need thereof.

In a specific embodiment, the present invention comprises use of acompound in a method of treatment of a condition caused or characterisedby excess body weight, e.g. the treatment and/or prevention of obesity,morbid obesity, morbid obesity prior to surgery, obesity linkedinflammation, obesity linked gallbladder disease, obesity induced sleepapnea, prediabetes, diabetes, esp. type 2 diabetes, hypertension,atherogenic dyslipidimia, atherosclerois, arteriosclerosis, coronaryheart disease, peripheral artery disease, stroke or microvasculardisease in an individual in need thereof.

In another aspect, the compounds described may be used in a method oflowering circulating LDL levels, and/or increasing HDL/LDL ratio.

In a specific embodiment, the present invention comprises use of acompound in a method of lowering circulating LDL levels, and/orincreasing HDL/LDL ratio in an individual in need thereof.

In another aspect, the compounds described may be used in a method oflowering circulating triglyceride levels.

Pharmaceutical Compositions

The compounds of the present invention may be formulated aspharmaceutical compositions prepared for storage or administration. Sucha composition typically comprises a therapeutically effective amount ofa compound of the invention, in the appropriate form, in apharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention, and may be confirmed in properly designed clinical trials.The compounds of the present invention may be particularly useful fortreatment of humans.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. The termfurther encompases any agents listed in the US Pharmacopeia for use inanimals, including humans.

The term “pharmaceutically acceptable salt” refers to a salt of any oneof the compounds of the invention. Salts include pharmaceuticallyacceptable salts such as acid addition salts and basic salts. Examplesof acid addition salts include hydrochloride salts, citrate salts andacetate salts. Examples of basic salts include salts where the cation isselected from alkali metals, such as sodium and potassium, alkalineearth metals, such as calcium, and ammonium ions ⁺N(R³)₃(R⁴), where R³and R⁴ independently designates optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substituted aryl, oroptionally substituted heteroaryl. Other examples of pharmaceuticallyacceptable salts are described in “Remington's Pharmaceutical Sciences”,17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company,Easton, Pa., U.S.A., 1985 and more recent editions, and in theEncyclopaedia of Pharmaceutical Technology.

“Treatment” is an approach for obtaining beneficial or desired clinicalresults. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures in certain embodiments. Thosein need of treatment include those already with the disorder as well asthose in which the disorder is to be prevented. By treatment is meantinhibiting or reducing an increase in pathology or symptoms (e.g. weightgain, hyperglycemia) when compared to the absence of treatment, and isnot necessarily meant to imply complete cessation of the relevantcondition.

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. In certain embodiments, packaged formsinclude a label or insert with instructions for use. Compositions may beformulated for any suitable route and means of administration.Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, rectal, nasal, topical (including buccaland sublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy.

Subcutaneous or transdermal modes of administration may be particularlysuitable for the compounds described herein.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the compound,increase bioavailability, increase solubility, decrease adverse effects,achieve chronotherapy well known to those skilled in the art, andincrease patient compliance or any combination thereof. Examples ofcarriers, drug delivery systems and advanced drug delivery systemsinclude, but are not limited to, polymers, for example cellulose andderivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylatepolymers, polylactic and polyglycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, liquid crystals and dispersions thereof,L2 phase and dispersions there of, well known to those skilled in theart of phase behaviour in lipid-water systems, polymeric micelles,multiple emulsions, self-emulsifying, self-microemulsifying,cyclodextrins and derivatives thereof, and dendrimers.

Combination Therapy

A compound or composition of the invention may be administered as partof a combination therapy with an agent for treatment of obesity,hypertension, dyslipidemia or diabetes.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus a compound or composition of the invention can further be used incombination with an anti-obesity agent, including but not limited to aGLP-1 (glucagon-like peptide 1) receptor agonist (e.g. as describedbelow), peptide YY or analogue thereof, neuropeptide Y (NPY) or ananalogue thereof, cannabinoid receptor 1 antagonist, lipase inhibitor,Human prolslet Peptide (HIP), melanocortin receptor 4 agonist, melaninconcentrating hormone receptor 1 antagonist, phentermine (alone or incombination with topiramate), a combination of norepinephrine/dopaminereuptake inhibitor and opioid receptor antagonist (e.g. a combination ofbupropion and naltrexone), Orlistat™, Sibutramine™, CCK, amylin,pramlintide and leptin, as well as analogues thereof or a serotonergicagent (e.g. lorcaserin).

A compound or composition of the invention can be used in combinationwith an anti-hypertension agent, including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretics, beta-blocker, or calcium channel blocker.

A compound or composition of the invention can be used in combinationwith a dyslipidaemia agent, including but not limited to a statin, afibrate, a niacin, a PSCK9 (Proprotein convertase subtilisin/kexin type9) inhibitor and/or a cholesterol absorption inhibitor.

Further, a compound or composition of the invention can be used incombination with an anti-diabetic agent, including but not limited to abiguanide (e.g. metformin), a sulfonylurea, a meglitinide or glinide(e.g. nateglinide), a DPP-IV inhibitor, an SGLT2 inhibitor, a glitazone,a GLP-1 receptor agonist (which is different from the compounds of theinvention), an SGLT2 inhibitor (i.e. an inhibitor of sodium-glucosetransport, e.g. a gliflozin such as empagliflozin, canagliflozin,dapagliflozin or ipragliflozin), a GPR40 agonist (FFAR1/FFA1 agonist,e.g. fasiglifam), or an insulin or an insulin analogue.

Examples of GLP-1 receptor agonists include GLP-1 and GLP-1 analogues,exendin-4 and exendin-4 analogues, liraglutide (Saxenda™, Victoza™),exenatide (Byetta™ and Bydureon™), Byetta LAR™, lixisenatide (Lyxumia™),Dulaglutide and Albiglutide.

Examples of insulin analogues include, but are not limited to, Lantus™,Novorapid™, Humalog™, Novomix™ Actraphane™ HM, Levemir™ Degludec™ andApidra™.

EXAMPLES Example 1: General Synthesis of Glucagon Analogues

Solid phase peptide synthesis (SPPS) was performed on a microwaveassisted synthesizer using standard Fmoc strategy in NMP on apolystyrene resin (TentaGel S Ram). HATU was used as coupling reagenttogether with DIPEA as base. Piperidine (20% in NMP) was used fordeprotection. Pseudoprolines: Fmoc-Phe-Thr(psiMe,Mepro)-OH (purchasedfrom NovaBiochem) was used as applicable.

Abbreviations employed are as follows:

-   Boc: tert-butyloxycarbonyl-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl-   DCM: dichloromethane-   DMF: N,N-dimethylformamide-   DIPEA: diisopropylethylamine-   EDT: 1,2-ethanedithiol-   EtOH: ethanol-   Et₂O: diethyl ether-   HATU:    N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium    hexafluorophosphate N-oxide-   MeCN: acetonitrile-   NMP: N-methylpyrrolidone-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane

Cleavage:

The crude peptide was cleaved from the resin by treatment with95/2.5/2.5% (v/v) TFA/TIS/water at room temperature (r.t.) for 2 hours.Most of the TFA was removed at reduced pressure and the crude peptidewas precipitated and washed with diethylether and allowed to dry toconstant weight at ambient temperature.

Compound 1:H-H-Aib-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH₂

Semaglutide and Liraglutide were used as reference compounds.

Example 2: Glucagon Receptor and GLP-1-Receptor Efficacy Assays

The cDNA encoding either the human glucagon receptor (Glucagon-R)(primary accession number P47871) or the human glucagon-like peptide 1receptor (GLP-1R) (primary accession number P43220) were synthesized andcloned into a mammalian expression vector containing a Zeocin resistancemarker.

The mammalian expression vectors encoding the Glucagon-R or the GLP-1-Rwere transfected into Chinese hamster ovary (CHO) cells by theAttractene method. Stably expressing clones were obtained by Zeocinselection (250 μg/mL) upon limited dilution of cells resistant to theselection pressure. Glucagon-R and GLP-1-R cell clones expressing werepicked, propagated and tested in the Glucagon-R and GLP-1-R efficacyassays as described below. One Glucagon-R expressing clone and oneGLP-1-R expressing clone were chosen for compound profiling.

CHO cells expressing the human Glucagon-R, or human GLP-1-R were seeded24 hours prior to the assay at 5000 cells per well in 384-wellmicrotiter plates in culture in 50 μl growth medium. On the day ofanalysis, growth medium was removed and the cells were washed once with100 μl of assay buffer (Krebs-Ringer-buffer; KRBH). The buffer wasremoved and the cells were incubated for 15 min at room temperature in10 μl KRBH (KRBH+10 mM HEPES, 5 mM NaHCO3, 0.1% (V/V) BSA) with 0.1 mMIBMX in deionized water containing increasing concentrations of testpeptides. The reaction was stopped by the addition of lysis buffer (0.1%w/v BSA, 5 mM HEPES, 0.3% v/v Tween-20). After cell lysis for 10 min atroom temperature, 10 μl of acceptor/donorbead mixture as contained inthe AlphaScreen™ cAMP Functional Assay Kit was added. After two hours ofincubation at room temperature in the dark, the cAMP content wasdetermined applying the AlphaScreen™ cAMP Functional Assay Kit fromPerkin-Elmer according to manufacturer instructions. EC₅₀ and relativeefficacies compared to reference compounds (glucagon and GLP-1) werecalculated applying computer aided curve fitting. The GLP-1/glucagonratio is calculated as defined earlier. See Table 1.

TABLE 1 EC50 EC50 hGCGR hGLP-1R Ratio Compound CHO-K1 [nM] CHO-K1 [nM]GLP-1/Glucagon 1 3.6 nM 0.48 nM 0.13

Example 3: Agonistic Activity on Endogenous GLP-1 Receptor

Agonistic activity of the test compounds on endogenous GLP-1 receptorswas determined using a murine insulinoma cell line. Intracellular cAMPwas used an indicator of receptor activation.

Cells were cultured for 24 h at a density of 10,000 cells/well in a384-well plate. Medium was removed and 10 μL KRBH buffer (NaCl 130 mM,KCl 3.6 mM, NaH₂PO₄ 0.5 mM, MgSO₄ 0.5 mM, CaCl₂ 1.5 mM) containing testcompound or GLP-1 (at increasing concentrations from 0.1 pM to 100 nM)or solvent control (0.1% (v/v) DMSO) was added to the wells for 15minutes at a temperature of 26° C.

The cellular cAMP content is measured using the AlphaScreen cAMPFunctional Assay Kit (Perkin Elmer). Measurement was performed using theEnvision (PerkinElmer) according to manufacturer's recommendations.

Results were converted into cAMP concentrations using a cAMP standardcurve prepared in KRBH buffer containing 0.1% (v/v) DMSO. The resultingcAMP curves were plotted as absolute cAMP concentrations (nM) over log(test compound concentration) and analyzed using the curve fittingprogram XLfit.

EC₅₀ was calculated as the concentration of test compound resulting in ahalf-maximal elevation of cAMP levels, reflecting the potency of thetest compound. See Table 2.

TABLE 2 Compound EC50 [nM] 1 0.54 nM

Example 4: Agonistic Activity on Endogenous Glucagon Receptor

Agonistic activity of the test compounds on endogenous glucagon receptorwas determined by measuring their effect on rate of glycogen synthesisin primary rat hepatocytes. Upon activation of the glucagon receptor, aninhibition of the glycogen synthesis rate is expected. Rate of glycogensynthesis was determined by counting the amount of radioactively labeledglucose incorporated into the cellular glycogen stores in a definedperiod of time.

Primary rat hepatocytes were cultured at a density of 40,000 cells/wellin a 24-well plate for 24 hours at 37° C. and 5% CO₂.

Medium was discarded and the cells washed with PBS. 180 μL of KRBH-basedbuffer containing 0.1% BSA and glucose at a concentration of 22.5 mM wasthen added to the wells, followed by test compound and 40 μCi/mlD-[U14C] glucose (20 μL each). Incubation was continued for 3 hours.

At the end of the incubation period, the incubation buffer was aspiratedand cells washed once with ice-cold PBS before lysis by incubation for30 min at room temperature with 100 μL 1 mol/NaOH.

Cell lysates were transferred to 96-well filter plates and glycogenprecipitated by incubating the filter-plates for 120 min at 4° C.followed by washing the filter plates 4 times with ice-cold ethanol(70%). The resulting precipitates were filtered to dryness and theamount of incorporated ¹⁴C-glucose determined by using a Topcountscintillation counter according to manufacturer's recommendations.

Wells with vehicle controls (0.1% (v/v) DMSO in KRBH buffer) wereincluded as reference for non-inhibited glycogen synthesis (100% CTL).Wells without added D-[U¹⁴C] glucose were included as controls fornon-specific background signal (subtracted from all values). Endogenousglucagon peptide was used as a positive control.

All treatments were performed at least in duplicate.

Parameters calculated to describe both the potency as well as theagonistic activity of each test compound on the endogenous glucagonreceptor are pEC50 and % CTL.

% CTL is determined by calculating the percentage of CPM/well in thepresence of the test compound compared to the CPM/well of the vehiclecontrol after subtracting the background CPM/well:[CPM/well(basal)−CPM/well(sample)]*100/[CPM/well(basal)−CPM/well(control)]

An activator of the glucagon receptor will result in an inhibition ofthe glycogen synthesis rate and will give % CTL values between 0% CTL(complete inhibition) and 100% CTL (no observable inhibition).

The resulting activity curves were plotted as absolute counts (unit:cpm/sample) over log (test compound concentration) and analyzed usingthe curve fitting program XLfit.

EC₅₀ was calculated as a measure of the potency of the test compound,and is shown in Table 3.

TABLE 3 Compound EC50 [nM] 1 5.6 nM

The terms EC₅₀ and pEC₅₀ quoted in relation to GLP-1R activation couldequally be regarded as IC₅₀ and pIC₅₀ in relation to glycogen synthesis.

Example 5: Estimate of Pharmacokinetic Parameters

Pharmacokinetic parameters of the test compounds were determined afterintravenous administration to Han/Wistar rats. The acylated GLP-1analogue semaglutide was also tested for comparison purposes.

Male Wistar rats were obtained from Charles River (Germany) weighingapproximately 180 to 210 g at time of arrival at the test facility. Ratswere caged in European standard rat cages type IV with light cycle of12-hour dark and 12-hour light. During the study rats were housed instandard rat cages type III. Both diet Altromin 1324 (Altromin, Germany)and water was administered ad libitum during the whole experimentalperiod. The animals were housed in the test facility for at least 4 daysin order to assure proper acclimatization.

The compounds were first dissolved in 0.1% aqueous ammonia to a nominalconcentration of 2 mg/ml, and then diluted to the desired dosingstrength (10 μM) in sterile PBS containing 25 mM phosphate buffer, pH7.4. Intravenous injections corresponding to 20 nmol/kg were given via alateral tail vein.

Blood samples (200 μl) were collected from the periorbital plexus attime points 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 32 and 48 h post dosinginto K₃EDTA tubes and centrifuged for 5 minutes at 4° C. within 20minutes of sampling. Plasma samples (>100 μl) were transferred to96-well PCR plates, immediately frozen and kept at −20° C. untilanalysed for plasma concentration for the respective GLP-1-glucagoncompound using LC-MS/MS. Individual plasma concentration-time profileswere analysed by a non-compartmental approach using ToxKin™ version 3.2(Unilog IT Services), and the resulting pharmacokinetic parameters weredetermined. See Table 4.

TABLE 4 Mean Clearance Terminal Residence Compound (ml/min/kg) half life(h) Time (h) 1 0.09 15.2 19.2 Semaglutide 0.10 9.0 11.4

Example 6: Oral Glucose Tolerance Test

Effects of the test compounds on glycemic control were determined by anoral glucose tolerance test (OGTT) in 7-8 weeks old male C57BL6/J mice(obtained from Charles River Laboratories, Germany). Animals were housedin groups in individually ventilated cages (Tecniplast green line) witha 12-hour light cycle. They had ad libitum excess to a standard rodentdiet (Provimi Kliba 3438) and water. The animals were housed in the testfacility for at least one week prior to the OGTT. All experimentalprotocols concerning the use of laboratory animals were reviewed by afederal Ethics Committee and approved by governmental authorities.

The compounds were first dissolved in 0.1% aqueous ammonia to a nominalconcentration of 2 mg/ml, and then diluted to the desired dosingstrength in sterile PBS containing 25 mM phosphate buffer, pH 7.4.Compounds were administered in the morning by subcutaneous injection ata dose of 30 nmol/kg and a volume of 5 ml/kg. Control animals received avehicle injection only. The GLP-1 analogue liraglutide was tested forcomparison purposes. Group size was 5 animals per group.

The OGTT was performed 24 h or 48 h after compound administration.Animals were fasted for 10 h before the OGTT but still had ad libitumexcess to water. After overnight fasting, a baseline blood sample (0min) was obtained by tail bleed and blood glucose was measured with aglucometer. The animals were then challenged with an oral glucose load(2 g/kg) given as solution by gavage (5 ml/kg). Additional blood samplesfor glucose measurement were obtained by tail bleed at serial timepoints after the glucose load (15, 30, 60, 90, and 120 min).

Glucose excursion was quantified by calculating the total area under theblood glucose-time curve (AUC) between 0 min and 120 min. Calculation ofAUC was done by the trapezoidal rule without baseline correction. Thedata are expressed as mean % of control (% CTL). A value of 100% CTLindicates no observable effect, and values statistically significantbelow 100% CTL show an improvement of glucose tolerance. See Table 5.

TABLE 5 OGTT AUC 30 nmol/kg [% CTL] 24 h 48 h Compound 1 56 74Liraglutide 73

The invention claimed is:
 1. A compound having the formula:R¹-H-Aib-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-R²  (SEQID NO: 1) wherein R¹ is H (hydrogen), C₁₋₄ alkyl, acetyl, formyl,benzoyl or trifluoroacetyl; and R² is OH or NH₂; or a pharmaceuticallyacceptable salt thereof.
 2. A compound according to claim 1 which is:H-H-Aib-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH₂  (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof.
 3. Acomposition comprising a compound according to claim 1 in admixture witha carrier.
 4. A composition according to claim 3 wherein the compositionis a pharmaceutical composition, and the carrier is a pharmaceuticallyacceptable carrier.
 5. A method of preventing weight gain or promotingweight loss in an individual in need thereof comprising administering aneffective amount of a compound according to claim
 1. 6. A method oflowering circulating LDL levels, and/or increasing HDL/LDL ratio in anindividual in need thereof comprising administering an effective amountof a compound according to claim
 1. 7. A method of improving glycemiccontrol or of prevention or treatment of obesity, morbid obesity, morbidobesity prior to surgery, obesity linked inflammation, obesity linkedgallbladder disease, obesity induced sleep apnea, diabetes, metabolicsyndrome, hypertension, atherogenic dyslipidemia, atherosclerosis,arteriosclerosis, coronary heart disease, peripheral artery disease,stroke or microvascular disease comprising administering an effectiveamount of a compound according to claim
 1. 8. The method according toclaim 5, wherein the compound is administered as part of a combinationtherapy together with an agent for treatment of diabetes, obesity,dyslipidemia or hypertension.
 9. The method according to claim 8 whereinthe agent for treatment of diabetes is a biguanide, a sulfonylurea, ameglitinide or glinide, a DPP-IV inhibitor, an SGLT2 inhibitor, aglitazone, a GLP-1 receptor agonist, an SGLT2 inhibitor, a GPR40agonist, or an insulin or an insulin analogue.
 10. The method accordingto claim 8, wherein the agent for treatment of obesity is a GLP-1receptor agonist, peptide YY or analogue thereof, neuropeptide Y (NPY)or an analogue thereof, cannabinoid receptor 1 antagonist, lipaseinhibitor, Human prolslet Peptide (HIP), melanocortin receptor 4agonist, melanin concentrating hormone receptor 1 antagonist,phentermine (alone or in combination with topiramate), a combination ofnorepinephrine/dopamine reuptake inhibitor and opioid receptorantagonist, Orlistat™, Sibutramine™, CCK, amylin, pramlintide and leptinand analogues thereof, or a serotonergic agent.
 11. The method accordingto claim 8 wherein the agent for treatment of hypertension is anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretic, beta-blocker, or calcium channel blocker.
 12. Themethod according to claim 8 wherein the agent for treatment ofdyslipidemia is a statin, a fibrate, a niacin, a PSCK9 (Proproteinconvertase subtilisin/kexin type 9) inhibitor and/or a cholesterolabsorption inhibitor.
 13. A therapeutic kit comprising a compoundaccording to claim
 1. 14. A method of synthesis of a compound accordingto claim 1, wherein the synthesis comprises the step of solid-phase orliquid-phase peptide coupling in a method for synthesizing saidcompound.
 15. A method of producing a compound according to claim 1, themethod comprising expressing a precursor peptide sequence from a nucleicacid construct that encodes the precursor peptide, recovering theexpression product, and modifying the precursor peptide to yield acompound according to claim 1.